The Study Of Three-type ELISAs For α-casein In Bovine Milk

α-casein, is the most important component of milk proteins in terms of nutritional andbiological properties,accounting for40%of total proteins.Because of the concentration of α-caseinin bovine milk is steady,it can be used to the detection index of determining milk adulterated ornot.The two commercial standard products are looked as the object of the study,one of them has ahigh molecular weight,the other one is an oligopeptide which consists of6amino acids.The female BALB/C mice were immunized with α-casein (macromolecular substance) byintraperitoneal injection.After the third injection, the titer of the serum were tested by indirectELISA, and mice with high serum immunogen titers were immunized for reinforcement3daysprior to cell fusion.The culture supernatants was tested by indirect ELISA,and the positive cloneswere obtained by limiting dilution until the positive percentage reached100%,named4H3,whichcan secrete antibody specially and stably.The subtype of the positive hybridoma cell lines wasIgG1.BALB/c female mice were injected intraperitoneal with the positive hybridoma cells to gainthe McAb.Through the Protein Affinity Chromatography, the McAb was purified.Then,NewZealand white rabbits were immunized at multiple s.c. sites with the standard α-casein.The bloodsamples were collected from the hearts of each rabbits,and stored at ultra-low temperature freezerafter a series of treatments.The characteristic of the McAb and the PcAb were analyzed.The molecular weigh of the oligopeptide is too small to motive animals output any antibodiesagainst the oligopeptide directly.The glutaraldehyde method was used to gain completedimmunogen (α-casein-BSA) and coating antigen (α-casein-OVA).New Zealand white rabbits wereimmunized at multiple s.c. sites with the completed immunogen (α-casein-BSA) to producepolyclonal antibody.Then blood samples were collected from the hearts of each rabbits,and storedat ultra-low temperature freezer after a series of treatments.In order to build a foundation of theimmunological detection methods, the characteristic of the PcAb was identified.The colloidal gold solution with20nanometers particle size was prepared by the reduction oftrisodium citrate,whose shape、size and distribution were determined by transmission electronmicroscopy.The commercial secondary antibody-HRP were linked with gold particles and the result was identified by transmission electron microscopy.In this study,there types of ELISA were founded:the first type is based on miscroplates.Astandard curve was obtained with sandwich ELISA.The regression equation of α-casein was y==1.3260x+6.1163with correlation coefficient R2=0.9934. The linear detection range was from0.1to10.0μg/mL,the minimum detection limit was found to be15ng/mL.The second type is based oncolloidal gold.The standard α-casein was disclosed by the sandwich ELISA,which combinedcolloidal gold and ELISA technology.The regression equation of α-casein was y=0.3012x+2.3311with correlation coefficient R2=0.9800. The linear detection range was from0.1to4μg/mL.The third type is based on PcAb of the oligopeptide.Through the standard oligopeptidewas disclosed by the indirect competitive ELISA, we got the regression equation: y=13.301x+91.457, with correlation coefficien R2=0.9930. The minimum detection limit of thethird type was about1ng/mL.And linear detection range was from from1ng/mL to2×106ng/mL.After comparing these three methods,the third one has a wider detection range,a lowerdetection limit while the detection takes a longer time than the other two types.